Secreted Proteins and Nucleic Acids Encoding Them

ABSTRACT

The invention involves a method of identifying nucleic acid sequences encoding signal peptide-containing proteins. The method features chimeric constructs containing a KRE9 gene that lacks a signal sequence. Yeast containing chimeric KRE9 plasmid constructs that encode signal sequences are selected based on their ability to grow on media in which sucrose is the sole carbon source.

BACKGROUND OF THE INVENTION

[0001] The invention relates to methods for identifying genes encodingsignal sequences.

[0002] The demonstrated clinical utility of certain growth factors andcytokines, for example, insulin, erythropoietin, granulocyte-colonystimulating factor, granulocyte-macrophage colony stimulating factor,human growth hormone, interferon-beta, and interleukin-2 in thetreatment of human disease has generated considerable interest inidentifying novel proteins of this class.

[0003] Since growth factors and cytokines are secreted proteins, theyoften possess “signal sequences” at their amino terminal end. The signalsequence directs a secreted or membrane protein to a sub-cellularmembrane compartment, the endoplasmic reticulum, from which the proteinis dispatched for secretion from the cell or presentation on the cellsurface. Techniques that detect signal sequences or nucleic acidsequences encoding a signal sequence have been employed as tools in thediscovery of novel cytokines and growth factors.

[0004] Among the methods that have been used co identify secretedproteins are methods that rely on the homology between some secretedproteins. For example, DNA probes or PCR oligonucleotides that recognizesequence motifs present in genes encoding known secreted proteins havebeen used in screening assays to identify novel secreted proteins. In arelated approach, homology-directed sequence searching of ExpressedSequence Tag (EST) sequences generated by high-throughput sequencing ofspecific cDNA libraries has been used to identify genes encodingsecreted proteins. Both of these approaches can identify a signalsequence when there is a high degree of similarity between the DNAsequence used as a probe and the putative signal sequence.

[0005] “Signal peptide trapping” has also been used to identify secretedproteins (Tashiro et al., 1993, Science 261:600-603; Honjo et al., 1996;U.S. Pat. No. 5,525,486, and U.S. Pat. No. 5,536,637). Generically, thistechnique involves the ligation of cDNA, prepared from various mRNAsources, to a reporter gene lacking a signal sequence. The resultingchimeric constructs are introduced into an appropriate host cell.Depending upon the nature of the reporter gene, host cells are scoredfor either the presence of reporter protein at the cell surface orsecretion of the reporter protein from cells. In both cases, a positivescore indicates that the cell harbors a chimeric construct having a cDNAencoding a signal sequence which directs the export of the reporterprotein to the cell surface or into the extracellular medium.

[0006] In a related method (Klein et al., 1996, Proc. Nat. Acad. Sci.USA 93:7108-7113; Jacobs, 1996, U.S. Pat. No. 5,536,637) theSaccharomyces cerevisiae gene, SUC2, which encodes a secreted invertaseprotein, is used as a reporter. Invertase catalyzes the hydrolysis ofsucrose into glucose and fructose, sugars which, unlike sucrose, can bereadily utilized by S. cerevisiae as a carbon source. Strains of S.cerevisiae that cannot secrete SUC2 protein are unable to grow on mediawith sucrose as the sole carbon source. Thus, a mutant SUC2 gene whichdoes not encode a signal peptide can be used as a reporter in signalsequence trapping. Chimeric constructs composed of random cDNAs fused toDNA encoding SUC2 lacking a signal sequence are transformed into S.cerevisiae, and transformants secreting chimeric SUC2 are selected bygrowing the transformants under conditions where sucrose is the solecarbon source. This method offers a genetic selection for cDNAs encodingsignal peptides.

SUMMARY OF THE INVENTION

[0007] The invention features a method for identifying nucleic acidsequences encoding signal sequences. Most secreted andmembrane-associated proteins possess such signal sequences composed of15-30 hydrophobic amino acid residues at their amino termini. Becausesignal sequences are present in secreted proteins andmembrane-associated proteins, the identified nucleic acid sequences,which will encode at least a portion of a secreted ormembrane-associated protein, can be used to isolate additional nucleicacid molecules encoding the entirety of the secreted ormembrane-associated protein.

[0008] KRE9 is an example of a yeast secreted protein. Yeast KRE9 nullmutants show severe growth retardation (essentially no growth) whenglucose is the sole carbon source. Growth of a KRE9 null mutant onglucose can be restored by transformation with DNA encoding wild typeKRE9 protein, but not by transformation with DNA encoding a mutant KRE9protein lacking a signal sequence. Thus, secretion of KRE9 protein viaits signal sequence is required for its normal function. Importantly,the presence of extracellular KRE9 protein does not rescue the KRE9 nullphenotype. This result suggests that KRE9 protein must pass through thesecretory pathway in order to exert its normal function. Although yeastKRE9 null mutants show essentially no growth when glucose is used as thecarbon source, they can be maintained on galactose because of inductionof the KNH1, a functional homolog of KRE9.

[0009] The invention features a method for identifying secreted andmembrane-associated proteins using yeast that lack functional KRE9protein and are transformed with a chimeric DNA molecule in which amutant KRE9 gene lacking its signal sequence encoding portion is fusedto a test sequence. The transformed yeast are grown on a selectivemedium that is designed permit (or prevent) growth of cells whichproduce functional, secreted KRE9. If the test sequence encodes a signalsequence (fused in-frame to the sequence encoding mature KRE9 protein),the yeast cell will grow (or not grow in the case of a selective mediumwhich is designed to prevent growth of cells expressing functional,secreted KRE9) on the selective medium. Thus, the invention features anovel selection method utilizing DNA constructs containing a chimericKRE9 gene in which the part of the KRE9 gene encoding the native KRE9signal sequence is replaced with a candidate signal sequence encodingsequence. The ability of these chimeric constructs to rescue KRE9 nullmutants grown on glucose is tested as follows. The chimeric constructsare used to transform KRE9 null mutants. The transformed cells aretransferred to plates having glucose as the sole carbon source. Thosechimeric constructs that allow a transformed KRE9 null mutant to grow onglucose contain candidate signal sequence encoding sequences.

[0010] Since growth factors and cytokines are secreted proteins,possessing signal sequences at their amino termini, signal sequencetrapping can be employed as a tool in the discovery of novel proteins ofthis class.

[0011] One embodiment of the methods of the invention includes thefollowing steps:

[0012] (a) obtaining a nucleic acid molecule which includes a chimericgene, the chimeric gene including a first portion and a second portion,the first portion encoding a KRE9 lacking a functional signal sequenceand the second portion being a heterologous nucleic acid sequence;

[0013] (b) transforming a yeast cell lacking a functional KRE9 gene withthe nucleic acid molecule; and

[0014] (c) determining whether the transformed yeast cell grows whensupplied with a medium that permits growth of a yeast cell expressingKRE9 having a functional signal sequence, but does not permit growth ofa yeast cell that does not express KRE9 having a functional signalsequence, wherein growth on the medium indicates that the heterologousnucleic acid sequence present in the yeast cell encodes a signalsequence.

[0015] In another embodiment the method, step (a) includes:

[0016] (i) obtaining double-stranded DNA; and

[0017] (ii) ligating the double-stranded DNA to a DNA molecule encodingKRE9 lacking a functional signal sequence to create a chimeric gene.

[0018] In another embodiment of the invention step (a) includes:

[0019] (i) obtaining double-stranded DNA;

[0020] (ii) ligating the double-stranded DNA to a DNA molecule encodingKRE9 lacking a functional signal sequence to create a chimeric gene;

[0021] (iii) transforming a bacterium with a nucleic acid molecule thatincludes the chimeric gene;

[0022] (iv) growing the transformed bacterium; and

[0023] (v) isolating the nucleic acid molecule which includes the achimeric gene from the transformed bacterium.

[0024] In another embodiment of the invention the method, in order toidentify the signal sequence, the method includes: isolating andsequencing a portion of the chimeric gene contained within a yeast cellthat grows when supplied with a medium that permits growth of a yeastcell expressing KRE9, but does not permit growth of a yeast cell thatdoes not express KRE9 having a functional signal sequence.

[0025] In various preferred embodiments, first portion of the nucleicacid molecule is pBOSS1; second portion of the nucleic acid molecule iscDNA; the yeast strain is Yscreen2; the medium contains glucose as thesole carbon source; the medium contains a calcineurin inhibitor; and themethod includes using a nucleic acid molecule encoding the signalsequence to screen an eukaryotic library for a full-length gene or cDNAencoding a protein comprising the identified signal sequence.

[0026] The invention also features a yeast cell transformed with anucleic acid molecule comprising a chimeric gene, the chimeric genecomprising a first portion and a second portion, the first portionencoding a KRE9 lacking a functional signal sequence and the secondportion being a heterologous nucleic acid sequence.

[0027] The invention also features a method that includes:

[0028] (a) obtaining a nucleic acid molecule which includes a chimericgene, the chimeric gene including a first portion and a second portion,the first portion encoding a KRE9 lacking a functional signal sequenceand the second portion being a heterologous nucleic acid sequence;

[0029] (b) transforming a yeast cell lacking a functional KRE9 gene withthe nucleic acid molecule; and

[0030] (c) determining whether the transformed yeast cell grows whensupplied with a medium that does not permit growth of a yeast cellexpressing KRE9 having a functional signal sequence, but does permitgrowth of a yeast cell that does not express KRE9 having a functionalsignal sequence, wherein lack of growth on the medium indicates that theheterologous nucleic acid sequence present in the yeast cell encodes asignal sequence. In a preferred embodiment the medium contains K1 killertoxin.

[0031] In another preferred embodiment step (a) includes: (i) obtaininga double-stranded DNA; and (ii) ligating the double-stranded DNA to aDNA molecule encoding KRE9 lacking a functional signal sequence tocreate a chimeric gene.

[0032] In a another preferred embodiment the method, in order toidentify the signal sequence, includes: isolating and sequencing aportion of the chimeric gene contained within the yeast cell that doesnot grow when supplied with a medium that does not permit growth of ayeast cell expressing KRE9, but does permit growth of a yeast cell thatdoes not express KRE9 having a functional signal sequence.

[0033] The invention also features the expression vector pBOSS-1 and agenetically engineered host cell which harbors pBOSS-1.

[0034] A “nonfunctional KRE9 gene” is a KRE9 gene having a mutation ordeletion in its signal sequence encoding portion such that the gene doesnot encode a functional signal sequence and thus does not produce afunctional KRE9 protein. Cells which fail to produce functional KRE9protein exhibit slow vegetative growth and are effectively unable togrow on glucose. In the case where the nonfunctional KRE9 gene isproduced by a point mutation, it is preferable that there be more thanone mutation to decrease the chance of reversion to the wild type.

[0035] The KRE9-based signal sequence trap of the invention includes apositive selection method to screen for putative signal sequenceencoding sequences. The selection strategy permits screening of a largenumber putative signal sequence encoding sequences because those cellsthat do not contain such a sequence essentially do not grow. This is incontrast to most other signal trap methods such as that described inU.S. Pat. No. 5,525,486 which rely solely on the detection of a proteinencoded by a reporter gene. Furthermore, because there is nocross-feeding, a relatively large number of yeast can screened on anygiven plate.

[0036] In an alternative selection method of the invention, a negativeselection is employed using K1 killer toxin. K1 killer toxin appears tokill sensitive yeast cells following binding to cell wall β1,6-glucans.Thus, cells with mutations in KRE9 are resistant to killing by K1 killertoxin. This selection method confers advantages similar those of thepositive selection strategy in that large numbers of putative signalsequence encoding sequences can be screened.

[0037] Without being bound by any particular theory, the KRE9 proteinreportedly encodes a soluble secretory-pathway protein required foryeast cell wall synthesis and growth. Specifically, the KRE9 proteinplays a significant role in synthesis of cell surface β1,6-glucan (Brownand Bussey, 1993, Mol. Cell. Biol. 13:6346-6356) which is necessary fornormal cell growth. When glucose is present in the medium, β1,6-glucansynthesis is normal provided that functional, secreted KRE9 protein ispresent. In the absence of functional KRE9 protein, yeast cells growslowly when glucose is provided in the medium because of abnormal cellwall synthesis.

[0038] The KRE9-based signal trap, which is based on biosyntheticrequirements, contrasts with the principle of signal trap systems basedon catabolic requirements, for example the SUC2 signal trap selectionsystem (U.S. Pat. No. 5,536,637). SUC2 protein is involved in catabolismin that it cleaves certain sugars to form nutrients which can be used asa carbon and energy source. As described above, the SUC2 signal trapselection system is based on the fact that yeast cells that lackfunctional SUC2 protein cannot utilize sucrose or raffinose as a carbonsource. Thus, SUC2 null cells cannot grow when sucrose or raffinose isthe sole carbon source.

[0039] One important advantage of a KRE9-based signal sequence trap ofthe invention is the low number of false positives generated by thismethod. This is in contrast to other signal trap methods such as thatbased on the yeast SUC2 (U.S. Pat. No. 5,536,637). SUC2 null mutants areunable to grow when the energy source is sucrose or raffinose. Whenpresented extracellularly, SUC2 protein can rescue SUC2 null mutantsgrown under restrictive conditions via a phenomenon referred to ascross-feeding. This arises because extracellular SUC2 protein cleavessucrose into diffusible nutrients on which neighboring yeast cells cangrow (i.e., fructose and glucose). KRE9 null mutants are not subject tocross-feeding, because extracellular KRE9 cannot restore growth of nullKRE9 mutants on glucose. Thus, a KRE9 gene engineered to lack its signalsequence can be used as a reporter in signal sequence trapping and willnot be subject to the background problems (i.e., false positives) thatlimit can limit the success of the less tightly regulated selectionsystems. Because the method of the invention is not subject tobackground problems to any significant degree, higher throughputscreening is possible.

[0040] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting. Other features and advantages of the invention will beapparent from the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0041]FIG. 1A is an illustration of the vector pBOSS1. FIG. 1B lists keysteps in the identification of cDNAs containing signal peptide encodingsequences.

[0042]FIG. 2 illustrates the nucleic acid sequence (SEQ ID NO:1) anddeduced amino acid sequence (SEQ ID NO:2) of exmos4all.

[0043]FIG. 3 illustrates the nucleic acid sequence (SEQ ID NO:3) anddeduced amino acid sequence (SEQ ID NO:4) of exmosb4f08.

[0044]FIG. 4 illustrates the deduced amino acid sequence of exmosb4all(SEQ ID NO:2) and its alignment (SEQ ID NO.14 to a portion of murinesemaphorin F (SEQ ID NO:5).

[0045]FIG. 5 illustrates the deduced amino acid sequence of exmosb4f08(SEQ ID NO:4) and its alignment (SEQ ID NO:15 to a portion of a putativecalcium binding protein (SEQ ID NO:6).

DETAILED DESCRIPTION

[0046] The present invention capitalizes on the S. cerevisiae redundantgene pair, KRE9 and KNH1 in a method for identifying signal sequenceencoding sequences and signal sequences. The KRE9 gene encodes asecreted protein, predicted to have a 21 amino acid signal peptide. TheKRE9 protein is required for the synthesis of the yeast cell wallpolymer (1→6)-β-glucan which makes up about half of the dry weight ofthe cell (Brown and Bussey, 1993, Mol. Cell. Biol. 13:6346-6356).Although KRE9 null strains grow very poorly on glucose (effectivelythere is no growth), these strains grow vigorously on galactose. This isprobably due to the induction by galactose of the KNH1 gene, afunctional homolog of the KRE9 gene with which it shares 46% identity(Dijkgraaf et al., 1996, Yeast 12:683-692). Thus, the KRE9 null strainsthat are an essential part of the invention can be maintained ongalactose, and selection for strains containing functional KRE9 can beperformed by selection on glucose.

[0047] KRE9 is used as a reporter in the signal sequence trap of theinvention. To use KRE9 as a reporter in signal sequence trapping, a KRE9null strain that is unable to grow under restrictive conditions (e.g.,when glucose is the sole carbon source provided in the medium) must beused. An example of a suitable KRE9 null strain (Yscreen2) is describedin Example 1. Other appropriate strains can be constructed using methodsdescribed in Example 1 and methods known to those in the art.

[0048] In one embodiment, the signal sequence trap of the inventioninvolves ligating a cDNA to a mutant KRE9 gene that does not encode asignal sequence, thus creating a chimeric gene (Example 1). The chimericgene is used to transform a yeast KRE9 null strain. The transformantsare then grown under a selective condition (e.g., in medium containingglucose as the sole carbon source) that does not permit growth of yeastthat are null for KRE9. Only those chimeric genes encoding a signalsequence can restore the function of KRE9 by facilitating its secretion,thus permitting growth under the selective condition (Example 2). Thisscreening strategy offers a rapid and efficient direct growth selectionfor cDNAs encoding a signal sequence and, as mentioned above, avoids theproblems of cross-feeding associated with the SUC2 method. The abilityof this method to identify novel sequences is demonstrated in Example 3.Various additional embodiments of the invention are described inExamples 4-5.

[0049] In one embodiment, the method of the invention includes thefollowing steps: a) obtain double-stranded cDNA from an eukaryotic celland ligate the eukaryotic cDNA to an appropriate plasmid vectorcontaining a mutant KRE9 gene that does not encode a signal sequence;then transform an E. coli with the ligated DNA, culture the transformedE. coli, and isolate plasmid DNA from the transformants; b) transform anS. cerevisiae KRE9 null mutant with the isolated plasmid DNA; and c)select transformed yeast strains encoding functional KRE9 fusionproteins by growth on a selective medium (e.g., glucose). The method canalso include the following additional steps: isolate plasmid DNA fromthe selected yeast; transform E. coli with the isolated DNA; isolateplasmid DNA from the transformed E. coli; determine the nucleotidesequence of the heterologous DNA; and analyze sequences to identifynovel secreted proteins.

[0050] A KRE9 nucleic acid for use in the invention can be obtained bycloning as described, for example in Brown and Bussey, 1993, supra. Thesequence of KRE9 is described in several databases including GenBank(Accession No. Z49449x1) and Swiss-Prot (Accession No. P39005.

[0051] A yeast expression vector appropriate for use in the inventioncan be constructed as described below (Example 1, step 2) or from othersuitable vectors. Examples of such vectors are described in, forexample, Pouwels et al. (Cloning Vectors, Elsevier, New York, 1987 andSupplements), Rose et al., 1990, Methods in Yeast Genetics: A LaboratoryCourse Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.; Guthrie and Fink, eds., 1991, Guide to Yeast Genetics andMolecular Biology, Meth. Enzymol. 194, Academic Press, Inc. Harcourt,Brace Jovanovich, N.Y., and athttp://bioinformatics.weizman.ac.il/bioscience/urllists/vector.htm, orhttp://vectordb.atcg.com/. An appropriate yeast expression vector foruse in the invention includes a suitable yeast promoter andtranscription terminator (e.g., those of alcohol dehydrogenase; ADH1),and a yeast origin of replication (e.g. the 2μ origin). For thoseembodiments including a selection step in E. coli; at least an E. coliorigin of replication, and one or more E. coli selectable markers suchas drug resistance genes (e.g., genes conferring ampicillin,chloramphenicol, or tetracycline resistance) are generally included inthe vector.

[0052] Although cDNA from any eukaryote can be used for the invention,in general, mammalian, preferably human cDNA is used. It is alsopossible to use genomic DNA instead of cDNA. Methods for inserting anucleic acid such as a cDNA into a yeast expression vector (plasmid)used in the invention are known in the art; including methods forobtaining cDNA, ligation of heterologous nucleic acids, transformationof yeast and bacteria, isolation of plasmids, and DNA sequencing andanalysis. The examples below describe acceptable methods for theseprocedures. Further guidance can be acquired from, for example, Ausubelet al., (Current Protocols in Molecular Biology, Green PublishingAssociates, Inc., and John Wiley & Sons, Inc., NY, 1989), Sambrook etal. (Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989), Guthrie and Fink(eds, Methods in Yeast Genetics: A Laboratory Course Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1991), and Guide toYeast Genetics and Molecular biology, Meth. Enzymol. 194, AcademicPress, Inc. Harcourt, Brace Jovanovich, N.Y.).

[0053] Nucleic acid sequences encoding putative signal sequences can beanalyzed using sequence analysis software such as the Sequence AnalysisSoftware Package of the Genetics Computer Group, University of WisconsinBiotechnology Center, 1710 University Avenue, Madison, Wis. 53705, withthe default parameters as specified therein. Parameters of a putativesignal sequence that can be measured using such software include theextent of homology to known sequences. The software package Signal P(Nielsen et al., 1997, Protein Engineering 10:1-6) can also be used toanalyze a signal sequence.

[0054] The invention also encompasses screening cDNA or genomiclibraries to obtain full-length cDNAs or genes using a nucleic acidencoding a signal sequence identified as described herein. Many suchlibraries are known in the art. Methods of constructing cDNA and genomiclibraries are known in the art (for example, see Sambrook et al., 1989,supra; Ausubel et al., 1989, supra).

[0055] A library is screened by hybridizing nucleic acid moleculesencoding sequences (identified as described above) to nucleic acidmolecules in a library under stringent conditions. The sequence encodinga signal sequence can also be used to identify sequences encodinghomologous polypeptides in other species. Accordingly, the inventionincludes methods of detecting and isolating these nucleic acidmolecules. Using these methods, a sample (for example, a nucleic acidlibrary, such as a cDNA or genomic library) is contacted (or “screened”)with a probe encoding at least a portion of an identified signalsequence that is at least 25 or 50 nucleotides long. The probeselectively hybridizes to nucleic acids encoding related polypeptides(or to complementary sequences thereof). The term “selectivelyhybridize” is used to refer to an event in which a probe binds tonucleic acids encoding the signal sequence (or to complementarysequences thereof) to a detectably greater extent than to nucleic acidsencoding other signal sequences (or to complementary sequences thereof).The probe, which can contain at least 25 (for example, 25, 50, 100, or200 nucleotides) can be produced using any of several standard methods(see, for example, Ausubel et al., “Current Protocols in MolecularBiology, Vol. I,” Green Publishing Associates, Inc., and John Wiley &Sons, Inc., NY, 1989). For example, the probe can be generated using PCRamplification methods in which oligonucleotide primers are used toamplify a signal sequence-specific nucleic acid sequence. The probes areused to screen a nucleic acid library, thereby detecting nucleic acidmolecules (within the library) that hybridize to the probe.

[0056] One single-stranded nucleic acid is said to hybridize to anotherif a duplex forms between them. This occurs when one nucleic acidcontains a sequence that is the reverse and complement of the other(this same arrangement gives rise to the natural interaction between thesense and antisense strands of DNA in the genome and underlies theconfiguration of the “double helix”). Complete complementarity betweenthe hybridizing regions is not required in order for a duplex to form;it is only necessary that the number of paired bases is sufficient tomaintain the duplex under the hybridization conditions used.

[0057] Typically, hybridization conditions are of low to moderatestringency. These conditions favor specific interactions betweencompletely complementary sequences, but also allows some non-specificinteraction between less than perfectly matched sequences. Afterhybridization, the nucleic acids can be “washed” under conditions ofmoderate or high stringency to dissociate duplexes that are boundtogether by some nonspecific interaction (the nucleic acids that formthese duplexes are thus not completely complementary).

[0058] As is known in the art, the optimal conditions for washing aredetermined empirically, often by gradually increasing the stringency.The parameters that can be changed to affect stringency include,primarily, temperature and salt concentration. In general, the lower thesalt concentration and the higher the temperature, the higher thestringency. Washing can be initiated at a low temperature (for example,room temperature) using a solution containing a salt concentration thatis equivalent to or lower than that of the hybridization solution.Subsequent washing can be carried out using progressively warmersolutions having the same salt concentration. As alternatives, the saltconcentration can be lowered and the temperature maintained in thewashing step, or the salt concentration can be lowered and thetemperature increased. Additional parameters can also be altered. Forexample, use of a destabilizing agent, such as formamide, alters thestringency conditions.

[0059] In reactions where nucleic acids are hybridized, the conditionsused to achieve a given level of stringency will vary. There is not oneset of conditions, for example, that will allow duplexes to form betweenall nucleic acids that are 85% identical to one another; hybridizationalso depends on unique features of each nucleic acid. The length of thesequence, the composition of the sequence (for example, the content ofpurine-like nucleotides versus the content of pyrimidine-likenucleotides) and the type of nucleic acid (for example, DNA or RNA)affect hybridization. An additional consideration is whether one of thenucleic acids is immobilized (for example, on a filter).

[0060] An example of a progression from lower to higher stringencyconditions is the following, where the salt content is given as therelative abundance of SSC (a salt solution containing sodium chlorideand sodium citrate; 2× SSC is 10-fold more concentrated than 0.2× SSC).Nucleic acids are hybridized at 42° C. in 2× SSC/0.1% SDS (sodiumdodecylsulfate; a detergent) and then washed in 0.2× SSC/0.1% SDS atroom temperature (for conditions of low stringency); 0.2× SSC/0.1% SDSat 42° C. (for conditions of moderate stringency); and 0.1× SSC at 68°C. (for conditions of high stringency). Washing can be carried out usingonly one of the conditions given, or each of the conditions can be used(for example, washing for 10-15 minutes each in the order listed above).Any or all of the washes can be repeated. As mentioned above, optimalconditions will vary and can be determined empirically.

[0061] Another example set of conditions that are considered “stringentconditions” are those in which hybridization is carried out at 50° C. inChurch buffer (7% SDS, 0.5% NaHPO₄, 1 M EDTA, 1% BSA), and washing iscarried out at 50° C. in 2× SSC.

[0062] Once detected, the nucleic acid molecules can be isolated andsequenced by any of a number of standard techniques (see, for example,Sambrook et al., “Molecular Cloning, A Laboratory Manual,” 2nd Ed. ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0063] Although ΔKRE9 function can be restored by heterologous mammaliansignal sequences, it is not clear whether all N-terminal protein fusionsof secreted proteins with ΔKRE9 will regain appropriate function. Forexample, fusion of KRE9 to a large portion of another protein mayinterfere with KRE9 function even under circumstances which permitsecretion of the fusion protein. This issue is addressed by theinclusion of a sequence encoding a cleavage site for the KEX2 protease(lysine-arginine-aspartic acid; Julius et al., 1984, Cell 37:1075) atthe junction between the mammalian cDNAs and the ΔKRE9 cDNA in thenucleic acid molecule of the invention (e.g., in the chimeric gene ofthe invention in pBOSS1). KEX2 can cleave the fusion protein as itpasses through the cellular secretory apparatus, thus relieving ΔKRE9 ofany functional impairment imposed by the N-terminal fusion.

EXAMPLES

[0064] The following examples illustrate the invention, includingconstructing an appropriate yeast strain and vector, and selectiveconditions useful for identifying a vector containing a sequenceencoding a signal sequence.

Example 1 Construction of a Signal Trap Screening System

[0065] Step 1: Strain Development

[0066] The first step in developing the KRE9-based signal peptidetrapping system was construction of an appropriate yeast strain.Standard media and techniques appropriate for Saccharomyces were used(Rose et al., 1990, Methods in Yeast Genetics: A Laboratory CourseManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;Guthrie and Fink, eds., 1991, Guide to Yeast Genetics and MolecularBiology, Meth. Enzymol. 194, Academic Press, Inc. Harcourt, BraceJovanovich, N.Y.). The parent strain used for the construction was thehaploid SEY 6210/kre9::HIS (mat a, leu2-3, ura3-52, his3-Δ200, lys2-801,trp-Δ901, suc2-Δ9) containing wild type KRE9 on a PRS 316/URA3 vector(Yscreen1; Brown and Bussey, 1993). This strain is maintained onSD/-his, -ura (1.7 g yeast nitrogen base without amino acids andammonium sulphate (DIFCO), 5 g ammonium sulfate, 0.66 g -His/-Uradropout powder (Clontech; Palo Alto, Calif.), 20 g dextrose, and 20 gBacto-Agar per liter).

[0067] Yscreen1 was further engineered to lose the PRS 316-KRE9Δcontaining plasmid, thereby creating a true KRE9 null strain. This wasaccomplished by plating Yscreen1 on SD/-his, replica plating ontoSD/-his containing 1 mg/ml 5-fluoroorotic acid (5-FOA, Sigma) and 20 g/lgalactose. These conditions select for the loss of the KRE9 wild-typeexpressed from the PRS316 and induces the KNH1 gene which functionallysubstitutes for the KRE9 gene. Colonies that grew on these plates werereplica plated onto SD/-his plus galactose and SD/-his/-ura plusglucose. Colonies which grow on SD/-his plus galactose and fail to growon SD/-his/-ura plus glucose are presumed to have lost the PRS 316-KRE9containing plasmid. A strain isolated in this way was designatedYscreen2. Saturated liquid cultures from a single colony were placed at−80° C. in 25% glycerol for long term storage.

[0068] Other disrupted strains of KRE9 that can be used in the inventioncan be constructed using methods known in the art (Baudin et al., 1994,Nuc. Acids Res. 21:3329-3330; Guthrie and Fink supra; Wach et al., 1994,Yeast 10:1793-1808).

[0069] Step 2: Plasmid Constructs

[0070] The plasmid vector used in the screen was constructed as follows.pACT2, a commercially available yeast expression vector (Clontech), wasdigested with Sma I and then partially digested with Hind III to removea 491 bp fragment containing the gal4 activation domain. The Hind IIIends were blunted with T4 DNA polymerase and the vector was religatedusing T4 DNA ligase. This vector designated pACT2-ΔH3/Sma.

[0071] The KRE9 gene was amplified out of S. cerevisiae usinggene-specific primers by PCR. For wild type KRE9 the 5′ primer was5′-CTCGAGCTCAGAGAATCAGCAACTGTGA-3′ (SEQ ID NO:7) and the 3′ primer was5′-AGATCTTCATACTTTTCTCATGTTGATTTTCC-3′ (SEQ ID NO:8). The resultingproduct has an Xho I site at the 5′ end and a Bgl II site at the 3′ end.This insert was cloned into pCR2.1 (Invitrogen; San Diego, Calif.).Individual colonies were sequenced to verify identity, digested with XhoI and Bgl II, and the KRE9 sequence ligated into pACT2-ΔH3/Sma to createpACT2-KRE9.

[0072] To generate a vector to be used for library construction, asimilar vector was prepared containing a KRE9 cDNA lacking the first 66nucleotides of KRE9 (ΔKRE9). These 66 nucleotides encode 22 amino acidsof a region which includes the translation initiation and predictedsignal peptide. Vector ΔKRE9 was amplified by PCR using the forwardprimer 5′-CTCGAGGTGAATATTGTTTCCCCCAGCTC-3′ (SEQ ID NO:9) and the same 3′primer as previously. This insert was cloned into pACT2-ΔH3/Sma to makepOSS1 (FIG. 1A). A third form of KRE9 (ΔKRE9met) containing aninitiating methionine codon but lacking a signal peptide was prepared ina similar manner, using the forward primer5-CTCGAGGATAATGGTGAATATTGTTTCCCCCAGCTC-3′ (SEQ ID NO:10) in combinationwith the same 3′ primer as before. The resulting cDNA was ligated intopACT2-ΔH3/Sma generating pACT2-ΔKRE9met. Finally, a DNA fragmentencoding the first 31 amino acids of human placental alkalinephosphatase (Genbank accession no. M13078; Millan, 1986, J. Biol. Chem.261:3112-3115, published erratum appears in J. Biol. Chem. 1991,266:4023), including signal sequence, was ligated in-frame to pBOSS1 asan EcoR I/Xho I fragment to generate a plasmid termed pBOSS-AP.

[0073] Step 3: Library Construction

[0074] cDNA for ligation to pBOSS1 was prepared from poly A+ RNAisolated from human osteoblasts by a modification of a commerciallyavailable cDNA synthesis kit (Stratagene: ZAP cDNA synthesis kit,catalog #200401). Single-stranded cDNA was synthesized from 5 μg ofhuman osteoblast polyA+ RNA using the following random hexamer primer(SEQ ID NO:11) incorporating an Xho I restriction site (underlined).

[0075] 5′-CTGACTCGAGNNNNNN-3′ (SEQ ID NO:11)

[0076] To generate short cDNA fragments, some of which would be expectedto represent the 5′ ends of mRNAs that contain signal sequences, randompriming was employed rather than the oligo d(T) priming method suggestedby Stratagene. The single-stranded cDNA was made double-stranded, DNAlinkers containing a free EcoR I overhang were ligated to both ends ofthe double-stranded cDNAs, and the linker-adapted double-stranded cDNAswere then digested with Xho I to generate a free Xho I overhang at the3′ ends of the cDNAs. All steps were performed using reagents from theStratagene ZAP cDNA synthesis kit according to the manufacturer'sinstructions. Linker-adapted double-stranded cDNAs were size selected bygel filtration through Sephacryl S-500 cDNA Size Fractionation Columns(Gibco BRL; Bethesda, Md.: Catalog #18092-015) according to themanufacturer's instructions.

[0077] Size selected, double-stranded cDNAs were ligated to pBOSS1 whichhad been digested with EcoR1 and Xho1 and purified by agarose gelelectrophoresis. Following overnight incubation at 16° C., the ligationreactions were extracted with phenol/chloroform and precipitated withthree volumes of absolute ethanol. Following centrifugation andextensive washing with 70% ethanol, the precipitate was resuspended in 5μl water, and 1 μl of the suspension was used to transformelectrocompetent DH10B E. coli (Gibco BRL) according to manufacturer'sinstructions using a Bio-Rad electroporation apparatus. Thetransformation was titered by plating dilutions of electroporatedbacteria on LB plates containing 100 μg/ml ampicillin. Once titered, theentire library was transformed, plated onto LB-ampicillin plates, andgrown overnight at 37° C. The following day, bacteria growing on theplates were scraped into LB, and plasmid DNA was prepared using Qiagenmega columns following manufacturer's instructions (Qiagen; SantaClarita, Calif.). DNA was quantitated spectrophotometrically andanalyzed by agarose gel electrophoresis.

[0078] Step 4: Yeast Transformation

[0079] To select and identify plasmids containing signal sequences (FIG.1B), a single colony of yeast strain Yscreen2 was inoculated into 50 mlof SC/-his/2% galactose and grown to saturation at 30° C. with shaking.This culture was diluted to an OD_(600nm) of 0.3 with fresh SC/-his/2%galactose, grown for approximately four hours to an OD_(600nm) of 0.8.The cells were collected by centrifugation, washed once with water, andresuspended in 1.5 ml TE/LiAc (10 mM Tris pH 8; 1 mM EDTA/100 mM lithiumacetate). To 50 μg of library DNA (see Example 3), 2 mg sonicatedherring testes DNA (Clontech: catalog #S0277; prepared by boiling for 20minutes and placing on ice for 5 minutes) and 1 ml Yscreen2 (prepared asabove) were added to a 50 ml conical tube. Six milliliters of PEG/LiAc(40% polyethylene glycol; LiAc (10 mM TRIS pH 8; 1 mM EDTA/100 mMlithium acetate) were added to tube and vortexed to mix. The mixture wasincubated at 30° C. for 30 minutes with shaking. Seventy microliters ofdimethylsulfoxide was added, the cells gently inverted to mix, and thenheat shocked for 15 minutes at 42° C., with occasional swirling. Cellswere pelleted, chilled on ice, and resuspended in 2.5 ml TE (10 mM TRISpH 8; 1 mM EDTA). Next, 250 μl of cells was plated onto each of ten15-cm plates containing selection media (SC/-his/-leu/2% glucose).Omitting histidine from growth plates maintains selection for disruptionof the endogenous KRE9 gene. Omitting leucine selects for the pBOSS1library plasmid, and the presence of glucose ensures that growth will beseen only in those cells having a functional signal peptide fused to theKRE9 polypeptide.

[0080] Plates were incubated for 2-4 days at 30° C. or until colonieswere apparent. Colonies were scraped from plates resuspended in 5 ml ofYPD, and pooled in a 50 ml conical tube. Next, the cells were pelleted,washed once with water, and resuspended in 1 ml yeast lysis buffer. Anequal volume of phenol:chloroform:isoamyl alcohol (25:24:1) and washedglass beads were added to tube containing yeast cells. The mixture wasvortexed vigorously for two minutes, spun in an Eppendorfmicrocentrifuge for 5 minutes, and the supernatant was transferred to aclean tube. To 40 μl DH10B electrocompetent cells, 0.5 μl of supernatant(as prepared above) was added, and mixed on ice. Cells wereelectroporated using a Bio-Rad Gene Pulser II system. One pulse wasdelivered at 2.5 kv, 25 μℑ, 100 Ωin a disposable electroporation cuvettewith a 0.1 cm gap (Bio-Rad; #165-2089). Following electroporation, 1 mlSOC was added, and the mixture was incubated with shaking at 30° C. for1 hour. Bacteria were plated on LB-ampicillin plates and incubatedovernight at 37° C. The next day, individual colonies were inoculatedinto 1 ml of LB-ampicillin culture medium in 96-well plates and grownovernight with shaking. One hundred microliter samples were transferredto a new 96-well plate containing 100 μl 50% glycerol per well, andstored at −80° C. A portion of glycerol stock was used to inoculatefresh LB-ampicillin cultures. Following overnight growth, an AGTC(Advanced Genetic Technology Corporation; Gaithersberg, Md.) plasmidpreparation was performed and the plasmids isolated from each culturewere sequenced from both ends to determine the presence and nature ofinserts. The forward sequencing primer was5′-GAGCAACGGTATACGGCCTTCCTT-3′ (SEQ ID NO:12), and the reversesequencing primer was 5′-GGGATATGCCCCATTATCCATC-3′ (SEQ ID NO:13).

Example 2 KRE9 Requires its Signal Sequence to Function and aHeterologous Mammalian Signal Peptide can Restore Function to KRE9Lacking its Native Signal Sequence

[0081] Various test constructs were used to transform the KRE9 nullmutant strain (Yscreen2). Expression vectors containing KRE9 with itssignal sequence removed (pACT-ΔKRE9), or containing KRE9 with its signalsequence removed but with a translation initiating methionine added(pACT-ΔKRE9met) were unable to rescue the growth of the KRE9 null mutanton glucose. Thus, cells expressing non-secretable KRE9 behave as nullmutants. In contrast, a vector containing a form of KRE9 in which thenative signal peptide was replaced with the signal peptide of humanplacental alkaline phosphatase (pBOSS-AP) did restore growth on glucose.These results indicate that KRE9 requires its signal sequence tofunction, and that heterologous mammalian signal peptides are able tosubstitute for the native KRE9 signal peptide. Thus, restoration offunction of an episomal non-secretable KRE9 gene can serve as the basisfor a screen for novel mammalian signal peptides in yeast.

Example 3 Screening of a Human Osteoblast cDNA Library Identifies NovelSignal Peptides

[0082] To identity novel mammalian signal peptides, a human osteoblastcDNA library was prepared in pBOSS1 and transformed into the yeaststrain Yscreen2 as described above. cDNA inserts of plasmids rescuedfrom the resulting yeast colonies after selection on glucose weresequenced. Of the novel signal peptides identified, two are representedin FIGS. 2 and 3.

[0083]FIG. 4 shows a 32 amino acid open reading frame, translated fromthe novel sequence identified in the cDNA clone shown in FIG. 2, termed1emxosb4a11, (SEQ ID NO:2) and its alignment (SEQ ID NO:14) with aprotein known as semaphorin F (SEQ ID NO:5). The sequence displays 68%identity (i.e., the aligned amino acid sequences are identical) and 81%similarity (i.e., the aligned amino acids are identical or areconservative changes) to the amino terminal signal peptide of semaphorinF (Genbank accession number X97817). Analysis of the emxosb4a11proteinsequence with the signal peptide prediction algorithm, Signal P (Nielsenet al., 1997, supra), confirmed that a likely cleavage site betweenamino acids 22 (a proline) and 23 (a glutamic acid) exists in this novelclone. Thus, clone emxosb4a11encodes the signal peptide of a novelprotein related to semaphorin F.

[0084]FIG. 5 shows a 108 amino acid open reading frame translated fromthe cDNA clone (FIG. 3) termed emxosb4f08 (SEQ ID NO:4), which fromamino acid 64 displays complete identity (SEQ ID NO:15) to the aminoterminal region of a putative calcium binding protein (SEQ ID NO:6)(Genbank accession number JS0027). Upstream of amino acid 64, however,the emxosb4f08 open reading frame extends to an initiating methioninewhich is followed by a stretch of hydrophobic residues characteristic ofa signal peptide. Analysis of this sequence with Signal P (Nielsen etal., 1997, supra) confirmed the presence of a signal peptide in thissequence, with a likely cleavage site between amino acids 24 (analanine) and 25 (a proline). Thus, emxosb4f08 encodes a novel form ofthis calcium binding protein which contains a signal sequence.

Example 4 Selection Using K1 Killer Toxin

[0085] KRE9 null mutants are resistant to the K1 killer toxin (Brown andBussey, 1993). The restoration of function of non-secretable KRE9 byheterologous signal sequences will restore toxin sensitivity. Thus,screening for colonies sensitive to K1 killer toxin offers asemiquantitative assay for KRE9 function.

[0086] K1 killer toxin is prepared using strain T158C/S14a as described(Bussey et al., 1983, Mol. Cell. Biol 3:1362-1370). Leu⁺ transformantscontaining library plasmids are tested by a zone of inhibition assay.For each strain, 0.1 ml of cell suspension (1×10⁷ cells/ml water) isadded to 10 ml of molten medium (e.g., 1% agar cooled to 45° C.containing either 1× Halverson's buffered YEPD, pH 4.7, or minimalmedium, pH 4.7). The agar-cell suspension is immediately poured intopetri plates. Concentrated toxin is spotted on the surface of thesolidified agar-cell suspension, and the plate is incubated overnight at18° C. followed by 24 hours at 30° C. Sensitive strains display a zoneof inhibition; the diameter of the zone is proportional to KRE9activity. Thus, resistant clones are eliminated from furtherconsideration while sensitive clones are prioritized by the diameter ofthe zone of inhibition.

Example 5 Selection in the Presence of Calcineurin Inhibition

[0087] KRE9 null mutants are hypersensitive to inhibitors of the proteinphosphatase calcineurin. This is because KNH1, which can functionallyreplace KRE9 when induced by galactose, is positively regulated bycalcineurin. Thus, calcineurin inhibitors, such as cyclosporin A andFK506, are expected to further suppress the KNH1 pathway onglucose-containing media, thereby increasing the likelihood that clonesgrowing on glucose contain a functional KRE9 chimera.

[0088] To use calcineurin in the selection of functional KRE9 chimeras,the growth of a Leu⁺ transformant containing a library plasmid iscompared to the growth of a control strain (e.g., Yscreen containingpACT2-KRE9) on solid medium (e.g., SD or YPD) containing a gradienteither FK506 (from 0 to 5 μg/ml) or cyclosporin A (from 0 to 100 μg/ml).Those strains that are as resistant to FK506 and/or cyclosporin A as thecontrol strain are prioritized for further analysis.

Other Embodiments

[0089] It is to be understood that while the invention has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention, which is defined by the scope of the appended claims.Other aspects, advantages, and modifications are within the scope of thefollowing claims.

1 15 1 517 DNA Homo Sapiens CDS (368)...(517) 1 ggggaccgtg tttgtggcccccaagccggt gccccccatt ttggaactca gcgagtaggg 60 ggcggctctg gggaagtggcagggggcgca gcagctgctg cctccacttc cctagccagg 120 tgctgaagag gatcttcggagccgctctgg cccccaggcg ctggatgact ggcaccagcg 180 ctcctcgcac ctgtgttggtgtgtgagact tgggctggag tgcccacgtg gctgtggagt 240 cagtgtgatt catgattgaggaaacgcgtc ctccatcctc tctctccttg gcactttcca 300 cacatgagga gaagaagagcttctgtttag aagacacgtg cccagagtca gaggcccctt 360 gcccacc atg aag gga acctgt gtt ata gca tgg ctg ttc tca agc ctg 409 Met Lys Gly Thr Cys Val IleAla Trp Leu Phe Ser Ser Leu 1 5 10 ggg ctg tgg aga ctc gcc cac cca gaggcc cag ggt acg act cag tgc 457 Gly Leu Trp Arg Leu Ala His Pro Glu AlaGln Gly Thr Thr Gln Cys 15 20 25 30 cag aga aca ctc gag gtg aat att gtttcc ccc agc tcc aag gca aca 505 Gln Arg Thr Leu Glu Val Asn Ile Val SerPro Ser Ser Lys Ala Thr 35 40 45 ttc agt cca agt 517 Phe Ser Pro Ser 502 50 PRT Homo Sapiens 2 Met Lys Gly Thr Cys Val Ile Ala Trp Leu Phe SerSer Leu Gly Leu 1 5 10 15 Trp Arg Leu Ala His Pro Glu Ala Gln Gly ThrThr Gln Cys Gln Arg 20 25 30 Thr Leu Glu Val Asn Ile Val Ser Pro Ser SerLys Ala Thr Phe Ser 35 40 45 Pro Ser 50 3 506 DNA Homo Sapiens CDS(132)...(506) 3 ttcttcctag tttctttttc ggcacaatat ttcaagttat accaagcatacaatcaactc 60 ccaagttggg atccgaattc ggcacgagcg gcacgagttg tgcttcggagaccgtaagga 120 tattgatgac c atg aga tcc ctg ctc aga acc ccc ttc ctg tgtggc ctg 170 Met Arg Ser Leu Leu Arg Thr Pro Phe Leu Cys Gly Leu 1 5 10ctc tgg gcc ttt tgt gcc cca ggc gcc agg gct gag gag cct gca gcc 218 LeuTrp Ala Phe Cys Ala Pro Gly Ala Arg Ala Glu Glu Pro Ala Ala 15 20 25 agcttc tcc caa ccc ggc agc atg ggc ctg gat aag aac aca gtg cac 266 Ser PheSer Gln Pro Gly Ser Met Gly Leu Asp Lys Asn Thr Val His 30 35 40 45 gaccaa gag cat atc atg gag cat cta gaa ggt gtc atc aac aaa cca 314 Asp GlnGlu His Ile Met Glu His Leu Glu Gly Val Ile Asn Lys Pro 50 55 60 gag gcggag atg tcg cca caa gaa ttg cag ctc cat tac ttc aaa atg 362 Glu Ala GluMet Ser Pro Gln Glu Leu Gln Leu His Tyr Phe Lys Met 65 70 75 cat gat tatgat ggc aat aat ttg ctt gat ggc tta gaa ctc tcc aca 410 His Asp Tyr AspGly Asn Asn Leu Leu Asp Gly Leu Glu Leu Ser Thr 80 85 90 gcc atc act catgtc cat aag gag gaa ggg agt gaa cag gca cca ctc 458 Ala Ile Thr His ValHis Lys Glu Glu Gly Ser Glu Gln Ala Pro Leu 95 100 105 gag gtg aat attgtt tcc ccc agc tcc aag gca aca ttc agt cca agt 506 Glu Val Asn Ile ValSer Pro Ser Ser Lys Ala Thr Phe Ser Pro Ser 110 115 120 125 4 125 PRTHomo Sapiens 4 Met Arg Ser Leu Leu Arg Thr Pro Phe Leu Cys Gly Leu LeuTrp Ala 1 5 10 15 Phe Cys Ala Pro Gly Ala Arg Ala Glu Glu Pro Ala AlaSer Phe Ser 20 25 30 Gln Pro Gly Ser Met Gly Leu Asp Lys Asn Thr Val HisAsp Gln Glu 35 40 45 His Ile Met Glu His Leu Glu Gly Val Ile Asn Lys ProGlu Ala Glu 50 55 60 Met Ser Pro Gln Glu Leu Gln Leu His Tyr Phe Lys MetHis Asp Tyr 65 70 75 80 Asp Gly Asn Asn Leu Leu Asp Gly Leu Glu Leu SerThr Ala Ile Thr 85 90 95 His Val His Lys Glu Glu Gly Ser Glu Gln Ala ProLeu Glu Val Asn 100 105 110 Ile Val Ser Pro Ser Ser Lys Ala Thr Phe SerPro Ser 115 120 125 5 32 PRT Mus musculus 5 Met Lys Gly Ala Cys Ile LeuAla Trp Leu Phe Ser Ser Leu Gly Val 1 5 10 15 Trp Arg Leu Ala Arg ProGlu Thr Gln Asp Pro Ala Lys Cys Gln Arg 20 25 30 6 45 PRT Homo Sapiens 6Met Ser Pro Gln Glu Leu Gln Leu His Tyr Phe Lys Met His Asp Tyr 1 5 1015 Asp Gly Asn Asn Leu Leu Asp Gly Leu Glu Leu Ser Thr Ala Ile Thr 20 2530 His Val His Lys Glu Glu Gly Ser Glu Gln Ala Pro Leu 35 40 45 7 28 DNAArtificial Sequence Primer 7 ctcgagctca gagaatcagc aactgtga 28 8 32 DNAArtificial Sequence Primer 8 agatcttcat acttttctca tgttgatttt cc 32 9 29DNA Artificial Sequence Primer 9 ctcgaggtga atattgtttc ccccagctc 29 1036 DNA Artificial Sequence Primer 10 ctcgaggata atggtgaata ttgtttcccccagctc 36 11 16 DNA Artificial Sequence Primer 11 ctgactcgag nnnnnn 1612 24 DNA Artificial Sequence Primer 12 gagcaacggt atacggcctt cctt 24 1322 DNA Artificial Sequence Primer 13 gggatatgcc ccattatcca tc 22 14 32PRT Homo Sapiens 14 Met Lys Gly Thr Cys Val Ile Ala Trp Leu Phe Ser SerLeu Gly Leu 1 5 10 15 Trp Arg Leu Ala His Pro Glu Ala Gln Gly Thr ThrGln Cys Gln Arg 20 25 30 15 108 PRT Homo Sapiens 15 Met Arg Ser Leu LeuArg Thr Pro Phe Leu Cys Gly Leu Leu Trp Ala 1 5 10 15 Phe Cys Ala ProGly Ala Arg Ala Glu Glu Pro Ala Ala Ser Phe Ser 20 25 30 Gln Pro Gly SerMet Gly Leu Asp Lys Asn Thr Val His Asp Gln Glu 35 40 45 His Ile Met GluHis Leu Glu Gly Val Ile Asn Lys Glu Ala Glu Met 50 55 60 Ser Pro Gln GluLeu Gln Leu His Tyr Phe Lys Met His Asp Tyr Asp 65 70 75 80 Gly Asn AsnLeu Leu Asp Gly Leu Glu Leu Ser Thr Ala Ile Thr His 85 90 95 Val His LysGlu Glu Gly Ser Glu Gln Ala Pro Leu 100 105

What is claimed is:
 1. A method comprising: (a) obtaining a nucleic acidmolecule comprising a chimeric gene, said chimeric gene comprising afirst portion and a second portion, the first portion encoding a KRE9lacking a functional signal sequence and the second portion being aheterologous nucleic acid sequence; (b) transforming a yeast celllacking a functional KRE9 gene with said nucleic acid molecule; and (c)determining whether said transformed yeast cell grows when supplied witha medium that permits growth of a yeast cell expressing KRE9 having afunctional signal sequence, but does not permit growth of a yeast cellthat does not express KRE9 having a functional signal sequence, whereingrowth on said medium indicates that said heterologous nucleic acidsequence present in said yeast cell encodes a signal sequence.
 2. Themethod of claim 1, wherein step (a) comprises: (i) obtainingdouble-stranded DNA; (ii) ligating said double-stranded DNA to a DNAmolecule encoding KRE9 lacking a functional signal sequence to create achimeric gene.
 3. The method of claim 1, wherein step (a) comprises: (i)obtaining double-stranded DNA; (ii) ligating said double-stranded DNA toa DNA molecule encoding KRE9 lacking a functional signal sequence tocreate a chimeric gene; (iii) transforming a bacterium with said nucleicacid molecule comprising a chimeric gene; (iv) growing said transformedbacterium; and (v) isolating said nucleic acid molecule comprising achimeric gene from said transformed bacterium.
 4. The method of claim 1,further comprising, in order to identify said signal sequence, isolatingand sequencing a portion of the chimeric gene contained within a yeastcell that grows when supplied with a medium that permits growth of ayeast cell expressing KRE9, but does not permit growth of a yeast cellthat does not express KRE9 having a functional signal sequence.
 5. Themethod of claim 1, wherein said second portion of said nucleic acidmolecule is pBOSS1.
 6. The method of claim 1, wherein said secondportion of said nucleic acid molecule is cDNA.
 7. The method of claim 1,wherein the yeast strain is Yscreen2.
 8. The method of claim 1, whereinsaid medium contains glucose as the sole carbon source.
 9. The method ofclaim 8, wherein the medium contains a calcineurin inhibitor.
 10. Themethod of claim 4, further comprising using a nucleic acid moleculeencoding said signal sequence to screen an eukaryotic library for afull-length gene or cDNA encoding a protein comprising said identifiedsignal sequence.
 11. A yeast cell transformed with a nucleic acidmolecule comprising a chimeric gene, said chimeric gene comprising afirst portion and a second portion, the first portion encoding a KRE9lacking a functional signal sequence and the second portion being aheterologous nucleic acid sequence.
 12. A method comprising: (a)obtaining a nucleic acid molecule comprising a chimeric gene, saidchimeric gene comprising a first portion and a second portion, the firstportion encoding a KRE9 lacking a functional signal sequence and thesecond portion being a heterologous nucleic acid sequence; (b)transforming a yeast cell lacking a functional KRE9 gene with saidnucleic acid molecule; and (c) determining whether said transformedyeast cell grows when supplied with a medium that does not permit growthof a yeast cell expressing KRE9 having a functional signal sequence, butdoes permit growth of a yeast cell that does not express KRE9 having afunctional signal sequence, wherein lack of growth on said mediumindicates that said heterologous nucleic acid sequence present in saidyeast cell encodes a signal sequence.
 13. The method of claim 12,wherein the medium contains K1 killer toxin.
 14. The method of claim 12,wherein step (a) comprises: (i) obtaining a double-stranded DNA; and(ii) ligating said double-stranded DNA to a DNA molecule encoding KRE9lacking a functional signal sequence to create a chimeric gene.
 15. Themethod of claim 12, further comprising, in order to identify said signalsequence, isolating and sequencing a portion of the chimeric genecontained within said yeast cell that does not grow when supplied with amedium that does not permit growth of a yeast cell expressing KRE9, butdoes permit growth of a yeast cell that does not express KRE9 having afunctional signal sequence.
 16. The expression vector pBOSS-1.
 17. Agenetically engineered host cell comprising the vector of claim 16.